Photoinduced hydrogen production by direct electron transfer from photosystem I cross-linked with cytochrome c3 to [NiFe]-hydrogenase.

The photosynthetic reaction center is an efficient molecular device for the conversion of light energy to chemical energy. In a previous study, we synthesized the hydrogenase/photosystem I (PSI) complex, in which Ralstonia hydrogenase was linked to the cytoplasmic side of Synechocystis PSI, to modify PSI so that it photoproduced molecular hydrogen (H2). In that study, hydrogenase was fused with a PSI subunit, PsaE, and the resulting hydrogenase-PsaE fusion protein was self-assembled with PsaE-free PSI to give the hydrogenase/PSI complex. Although the hydrogenase/PSI complex served as a direct light-to-H2 conversion system in vitro, the activity was totally suppressed by adding physiological PSI partners, ferredoxin (Fd) and ferredoxin-NADP+-reductase (FNR). In the present study, to establish an H2 photoproduction system in which the activity is not interrupted by Fd and FNR, position 40 of PsaE from Synechocystis sp. PCC6803, corresponding to the Fd-binding site on PSI, was selected and targeted for the cross-linking with cytochrome c3 (cytc3) from Desulfovibrio vulgaris. The covalent adduct of cytc3 and PsaE was stoichiometrically assembled with PsaE-free PSI to form the cytc3/PSI complex. The NADPH production by the cytc3/PSI complex coupled with Fd and FNR decreased to approximately 20% of the original activity, whereas the H2 production by the cytc3/PSI complex coupled with hydrogenase from Desulfovibrio vulgaris was enhanced 7-fold. Consequently, in the simultaneous presence of hydrogenase, Fd, and FNR, the light-driven H2 production by the hydrogenase/cytc3/PSI complex was observed (0.30 pmol Hz/mg chlorophyll/h). These results suggest that the cytc3/PSI complex may produce H2 in vivo.

Role of the aromatic ring of Tyr43 in tetraheme cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F.

Tyrosine 43 is positioned parallel to the fifth heme axial ligand, His34, of heme 1 in the tetraheme cytochrome c(3). The replacement of tyrosine with leucine increased the redox potential of heme 1 by 44 and 35 mV at the first and last reduction steps, respectively; its effects on the other hemes are small. In contrast, the Y43F mutation hardly changed the potentials. It shows that the aromatic ring at this position contributes to lowering the redox potential of heme 1 locally, although this cannot be the major contribution to the extremely low redox potentials of cytochrome c(3). Furthermore, temperature-dependent line-width broadening in partially reduced samples established that the aromatic ring at position 43 participates in the control of the kinetics of intramolecular electron transfer. The rate of reduction of Y43L cytochrome c(3) by 5-deazariboflavin semiquinone under partially reduced conditions was significantly different from that of the wild type in the last stage of the reduction, supporting the involvement of Tyr43 in regulation of reduction kinetics. The mutation of Y43L, however, did not induce a significant change in the crystal structure.

High-resolution wide-angle X-ray scattering of protein solutions: effect of beam dose on protein integrity.

Wide-angle X-ray scattering patterns from proteins in solution contain information relevant to the determination of protein fold. At relevant scattering angles, however, these data are weak, and the degree to which they might be used to categorize the fold of a protein is unknown. Preliminary work has been performed at the BioCAT insertion-device beamline at the Advanced Photon Source which demonstrates that one can collect X-ray scattering data from proteins in solution to spacings of at least 2.2 A (q = 2.8 A(-1)). These data are sensitive to protein conformational states, and are in good agreement with the scattering predicted by the program CRYSOL using the known three-dimensional atomic coordinates of the protein. An important issue in the exploitation of this technique as a tool for structural genomics is the extent to which the high intensity of X-rays available at third-generation synchrotron sources chemically or structurally damage proteins. Various data-collection protocols have been investigated demonstrating conditions under which structural degradation of even sensitive proteins can be minimized, making this technique a viable tool for protein fold categorization, the study of protein folding, unfolding, protein-ligand interactions and domain movement.

Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation.

Ultraviolet (UV) irradiation of HeLa cells triggers an apoptotic response mediated by mitochondria. Biochemical analysis of this response revealed that the elimination of cytosolic inhibitors is required for mitochondrial release of cytochrome c and subsequent caspase activation. These inhibitors were found to be Mcl-1 and Bcl-xL, two antiapoptotic members of the Bcl-2 family. Following UV treatment, Mcl-1 protein synthesis is blocked, the existing pool of Mcl-1 protein is rapidly degraded by the proteasome, and cytosolic Bcl-xL translocates to the mitochondria. These events are sequential; the elimination of Mcl-1 is required for the translocation of Bcl-xL. The disappearance of Mcl-1 is also required for other mitochondrial apoptotic events including Bax translocation, cytochrome c release, and caspase activation.

The tyrosine kinase Lck is involved in regulation of mitochondrial apoptosis pathways.

The induction of apoptosis requires the activation of a highly coordinated signaling network ultimately leading to the activation of caspases. In previous experiments we and others have shown that the tyrosine kinase Lck is required for adequate apoptosis induction in response to ionizing radiation, ceramide incubation and overexpression of the HIV-TAT protein. However, the position of Lck within given apoptotic signaling cascades remains unclear. We therefore aimed to define the role of Lck during radiation-induced apoptosis. Apoptosis induction in response to ionizing radiation, CD95 or TRAIL receptor stimulation was determined in Jurkat T-cells, the Lck-deficient Jurkat clone JCaM1.6- and Lck-retransfected JCaM1.6/Lck. No apoptosis, release of cytochrome c, breakdown of the mitochondrial potential were detectable during the first 48 h after irradiation of JCaM1.6 cells. In parallel, no activation of caspase-9, -8 and -3 was detectable. Since mitochondrial apoptosis pathways act within a feedback mechanism during death-receptor-mediated apoptosis, the influence of the Lck defect on CD95/Fas/Apo-1-L or TRAIL-induced apoptosis was also tested. Both stimuli induced apoptosis in Lck-deficient cells. However, the kinetics of apoptosis induction determined by caspase-8, -9 and -3 activation as well as deltapsi(m) breakdown was slowed. We conclude that the Lck deficiency influences early steps during radiation-induced mitochondrial alterations.

DNA damage, death receptor activation and reactive oxygen species contribute to ultraviolet radiation-induced apoptosis in an essential and independent way.

Nuclear DNA damage and death receptor (CD95) activation by ultraviolet-B radiation (UVB) play a major role in UVB-induced apoptosis. Removal of DNA damage combined with inhibition of death receptor activation resulted in pronounced but not complete suppression of apoptosis, indicating that a third independent pathway is involved. Since reactive oxygen species (ROS) cause apoptosis and are induced by UVB, the radical scavenger pyrrolidene-dithiocarbamate (PDTC) was used. PDTC prevented UVB-induced apoptosis partially, H(2)O(2)-induced cell death largely, but not CD95-mediated apoptosis. The same was observed for cytochrome c release from mitochondria, another important event during apoptosis. The proapoptotic protein Bid was cleaved upon exposure to UVB or to agonistic anti-CD95-antibodies, but not to H(2)O(2), indicating that H(2)O(2) uses a different pathway. The fact that PDTC neither inhibited CD95-mediated apoptosis nor affected UV-induced DNA damage indicated that ROS generated during UVB irradiation may directly trigger mitochondrial cytochrome c release, thereby contributing to apoptosis. Accordingly, complete inhibition of apoptosis was observed when in addition to DNA damage removal via photoreactivation and blockade of CD95 signaling by caspase-8 inhibitor zIETD, PDTC was added before UVB exposure. This indicates that DNA damage, death receptor activation and ROS formation contribute to UVB-induced apoptosis in an essential and independent way.

Optical study of cytochrome cM formation in Synechocystis.

The expression of the cytM gene, which encodes cytochrome cM in Synechocystis sp. PCC 6803, is induced under stress conditions such as low temperature. Results of spectrophotometric studies revealed that cytochrome cM was oxidized by light absorbed by photosystem I in cells that had acclimatized to a low temperature. However, no similar light-induced oxidation of cytochrome cM was observed in delta cytM mutant cells. The kinetics of the oxidation and reduction of P700 before and after acclimation of Synechocystis cells to low temperature suggested that cytochrome cM might donate electrons to photooxidized P700. Our observations indicate that cytochrome cM is synthesized under low-temperature stress and that it might carry electrons directly to P700+ in photosystem I.

Radiation induced cytochrome c release causes loss of rat colonic fluid absorption by damage to crypts and pericryptal myofibroblasts.

BACKGROUND: Therapeutic or accidental exposure to radiation commonly causes gastrointestinal disturbances, including diarrhoea. Rats subjected to whole body ionising radiation at a dose of 8 Gy lose their capacity to absorb fluid via the descending colon after four days. After seven days, fluid absorption recovers to control levels. AIMS: To investigate the effect of ionising radiation on colonic permeability together with its effect on mitochondria dependent apoptotic signals and intercellular adhesion molecules. METHODS: Rats were irradiated with doses of 0-12 Gy. Colonic permeability was measured by accumulation of fluorescein isothiocyanate (FITC) dextran in crypt lumens. Changes in levels of cytochrome c, caspase 3, E and OB cadherin, beta-catenin smooth muscle actin, and collagen IV were assessed using immunocytochemistry with confocal microscopy. RESULTS: Cytosolic cytochrome c increased after 8 Gy (t(1/2) 1.4 (0.6) hours) and peaked at approximately six hours. Caspase 3 increased more slowly, particularly in crypt epithelial cells (t(1/2) 57 (14.5) hours). Pericryptal myofibroblasts disintegrated within 24 hours as was evident from loss of OB cadherin and smooth muscle actin. This coincided with increased crypt permeability to dextran. Intercellular adhesion between crypt luminal cells was not lost until day 4 when both beta-catenin and E-cadherin were minimal. The half maximal dose-response for these effects was in the range 2-4 Gy. Recovery of colonic transport was concurrent with recovery of pericryptal smooth muscle actin and OB cadherin. The pan caspase inhibitor Z-Val-Ala-Asp.fluoromethylketone (1 mg/kg per day) had a small effect in conserving the pericryptal sheath myofibroblasts and sheath permeability but had no systemic therapeutic effects. CONCLUSIONS: These data suggest that radiation damage to the colon may be initiated by mitochondrial events. Loss of crypt fluid absorption and increased permeability coincided with decreased intercellular adhesion between crypt epithelial cells and loss of pericryptal sheath barrier function.

Protein kinase C inhibitor and irradiation-induced apoptosis: relevance of the cytochrome c-mediated caspase-9 death pathway.

Caspases are a family of cysteine proteases that constitute the apoptotic cell death machinery. We report the importance of the cytochrome c-mediated caspase-9 death pathway for radiosensitization by the protein kinase C (PKC) inhibitors staurosporine (STP) and PKC-412. In our genetically defined tumor cells, treatment with low doses of STP or the conventional PKC-specific inhibitor PKC-412 in combination with irradiation (5 Gy) potently reduced viability, enhanced mitochondrial cytochrome c release into the cytosol, and specifically stimulated the initiator caspase-9. Whereas treatment with each agent alone had a minimal effect, combined treatment resulted in enhanced caspase-3 activation. This was prevented by broad-range and specific caspase-9 inhibitors and absent in caspase-9-deficient cells. The tumor suppressor p53 was required for apoptosis induction by combined treatment but was dispensable for dose-dependent STP-induced caspase activation. These results demonstrate the requirement for an intact caspase-9 pathway for apoptosis-based radiosensitization by PKC inhibitors and show that STP induces apoptosis independent of p53.

Correlation of empirical magnetic susceptibility tensors and structure in low-spin haem proteins.

Experimental magnetic susceptibility tensors are reported for eight haems c with bis-His coordination. These data, obtained by fitting the dipolar shifts of backbone protons in the tetrahaem cytochromes c(3) from Desulfovibrio vulgaris and D. gigas, are analysed together with published values for other haem proteins. The x and y axes are found to rotate in the opposite sense to the axial ligands and are also counter-rotated with respect to the frontier molecular orbitals of the haem. The magnetic z-axis is close to the normal to the haem plane in each case. The magnitudes of the magnetic anisotropies are used to derive crystal field parameters and the rhombic splitting, V, is correlated with the dihedral angle between the axial ligands. Hence, it is apparent that the axial ligands are the dominant factor in determining the variation in magnetic properties between haems, and it is confirmed that "high g(max)" EPR signals are a reliable indicator of near-perpendicular ligands. These results are in full agreement with the analysis of non-Curie effects and electronic structure in the His-Met coordinated cytochromes c and C(551). Collectively, they show that the orientations of axial ligands to the haem may be estimated from single-crystal EPR data, from (13)C NMR shifts of the haem substituents, or from NMR dipolar shifts of the polypeptide.

Hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3.

The release of mitochondrial cytochrome c by genotoxic stress induces the formation of a cytosolic complex with Apaf-1 (mammalian CED4 homolog) and thereby the activation of procaspase-3 (cas-3) and procaspase-9 (cas-9). Here we demonstrate that heat-shock protein 27 (Hsp27) inhibits cytochrome c (cyt c)-dependent activation of cas-3. Hsp27 had no effect on cyt c release, Apaf-1 and cas-9 activation. By contrast, our results show that Hsp27 associates with cas-3, but not Apaf-1 or cas-9, and inhibits activation of cas-3 by cas-9-mediated proteolysis. Furthermore, the present results demonstrate that immunodepletion of Hsp27 depletes cas-3. Importantly, treatment of cells with DNA damaging agents dissociates the Hsp27/cas-3 complex and relieves inhibition of cas-3 activation. These findings define a novel function for Hsp27 and provide the first evidence that a heat shock protein represses cas-3 activation.

Dark aerobic growth conditions induce the synthesis of a high midpoint potential cytochrome c8 in the photosynthetic bacterium Rubrivivax gelatinosus.

In several strains of the photosynthetic bacterium Rubrivivax gelatinosus, the synthesis of a high midpoint potential cytochrome is enhanced 4-6-fold in dark aerobically grown cells compared with anaerobic photosynthetic growth. This observation explains the conflicting reports in the literature concerning the cytochrome c content for this species. This cytochrome was isolated and characterized in detail from Rubrivivax gelatinosus strain IL144. The redox midpoint potential of this cytochrome is +300 mV at pH 7. Its molecular mass, 9470 kDa, and its amino acid sequence, deduced from gene sequencing, support its placement in the cytochrome c8 family. The ratio of this cytochrome to reaction center lies between 0.8 and 1 for cells of Rvi. gelatinosus grown under dark aerobic conditions. Analysis of light-induced absorption changes shows that this high-potential cytochrome c8 can act in vivo as efficient electron donor to the photooxidized high-potential heme of the Rvi. gelatinosus reaction center.

[Effect of weak combined low frequency constant and alternative magnetic fields on intrinsic fluorescence of proteins in aqueous solutions]. / Vliianie slabykh kombinirovannykh postoiannogo i peremennogo magnitnykh polei na sobstvennuiu fluirestsentsiiu riada belkov v vodnykg rastvorakh.

It was shown that weak combined static (42 microT) and low-frequency variable (40 nT; 3-5 Hz) magnetic fields change the intensity of intrinsic fluorescence of some proteins (cytochrome c, bovine serum albumin, horseradish peroxidase, alkaline phosphatase). The effect can be interpreted as a change in the conformational state of the protein in water environment by the action of weak magnetic fields. The dynamics of the process, the concentration dependence, the binding of proteins to the fluorescence probe 1,8-ANS after treatment with magnetic fields, the frequency dependence of these reactions, and the dependence of the effect on the presence of the static constituent of the magnetic field were studied. It was shown that the changes in the intrinsic fluorescence of some enzymes (horseradish peroxidase, alkaline phosphatase) are related to changes in their functional activity. It was found that the effect is partially transferred via a solvent (water, 0.01 M NaCl) preliminarily treated with magnetic field. In the solvent, changes in its intrinsic fluorescence by the action of weak magnetic fields were also registered.

Photoionization of ferrocytochrome c by 248 nm laser light and the observation of the early stages of ferricytochrome c unfolding in the nanosecond-to-millisecond timescale.

Photolysis of ferrocytochrome c by 248 nm laser light in aqueous solution at pH 7 generates hydrated electrons (eaq-) by a monophotonic process with quantum yield phi = 0.034. Approximately three-quarters of the eaq- originate from the heme, which is converted from the ferrous to the ferric state in < 100 ns. The conformational changes associated with the change in the redox state of cytochrome c are either not detectable spectrophotometrically or complete in < 100 ns. Also, under conditions where ferrocytochrome c is stable but ferricytochrome c is unfolded (3 M guanidine, pH 7, 40 degrees C), photoionization of ferrocytochrome c generated ferricytochrome c with similar quantum yield. Under these conditions, the lifetime of native ferricytochrome c is 67 microseconds; it decays via two intermediates with lambda max > 410 nm, neither of which is the thermodynamically favored, unfolded form. These species are putatively identified as unfolding intermediates with nonnative iron ligands, similar to those found during folding of ferrocytochrome c. The results suggest that unfolding, like folding, proceeds by intrachain diffusion and ligand exchange.

In bacterial reaction centers, a key residue suppresses mutational blockage of two different proton transfer steps.

In reaction centers of Rhodobacter (Rb.) capsulatus, the M43Asn --> Asp substitution is capable of restoring rapid rates for delivery of the second proton to QB in a mutant that lacks L212Glu. Flash-induced absorbance spectroscopy was used to show a nearly native rate for transfer of the second proton to QB (approximately 700 s-1) in the L212Gln+M43Asp double-mutant reaction center; this rate was shown to decrease more than 1000-fold in the photoincompetent L212Glu --> Gln mutant [Miksovska, J., Kálmán, L., Maróti, P., Schiffer, M., Sebban, P., and Hanson, D.K. (1997) Biochemistry 36, 12216-12226]. In Rb. sphaeroides, the equivalent M44Asn --> Asp mutation was reported to restore the rate of transfer of the first proton to a wild-type level when it is added to the L213Asp --> Asn photoincompetent mutant [Rongey, S.H., Paddock, M.L., Feher, G., and Okamura, M.Y. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1325-1329]. It is remarkable that the same second-site mutation can compensate for both of these mutations which severely impair reaction center function by blocking two different proton-transfer reactions. We suggest that residue M43Asp is situated in a key position which can link pathways for delivery of both the first and second protons (involving structured water molecules) to QB.

Oxidation of ferric-cytochrome c by N, N'-bis (2-hydroperoxy-2-methoxyethyl)-1,4,5,8-naphthalene tetracarboxylic diimide, NP III--site specific modification of cytochrome c.

When the synthetic hydroxyl radical generator, N, N'-bis (2-Hydroperoxy-2-methoxyethyl)-1,4,5,8-naphthalene-tetracarboxylic diimide, NP III, was photoirradiated in the presence of ferric-cytochrome c (Fe3+), the fragmentation peak corresponding to the specific oxidative modification at the histidine site of cytochrome c was observed in the Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectroscopy (MALDI-TOF) measurement. Without photoirradiation, no such protein oxidation was observed in the experimental conditions employed. This result suggests the possible usage of NP-III as an oxidative protein-modifying reagent.

Oxidation of Fe(II) horse heart cytochrome c by ultrasound waves.

In aqueous solution, acoustically induced cavitation produces the collapse of bubbles containing gas and water-vapor, producing free radicals by the homolysis of the water molecules. Generally, under these extreme physical conditions, the secondary and tertiary structures of the proteins result are altered and denaturation phenomena are often observed. This paper discusses the evidence that, in the presence of argon and in oxygen-free experimental environment, the reduced horse heart cytochrome c, instead of undergoing a denaturation process, is oxidized to ferric-cytochrome c. Kinetic and circular dichroism measurements performed after ultrasound irradiation at a frequency of 38 kHz are reported. A possible correlation between ultrasound induced molecular damage to the tertiary structure of the proteins and their own extension of helix content is also hypothesized.

Membrane-bound c-type cytochromes in Heliobacillus mobilis. In vivo study of the hemes involved in electron donation to the photosynthetic reaction center.

The amount of heme per photosynthetic reaction center (RC) was examined in whole cells of Heliobacillus mobilis, and a stoichiometry of 5-6 hemes c and 1-3 hemes b per RC was found. Virtually the full complement of heme was seen to be functionally connected to the pool of electron donors to the photosynthetic RC. The kinetic parameters of electron transfer between reduced c-type hemes and the photooxidized primary donor P798+ were studied in whole cells and membrane fragments. The in vivo half-times of electron donation (50% with t 1/2 = 110 microseconds, 50% with t 1/2 = 600 microseconds) were seen to slow down to half-times in the range of several and several tens of milliseconds following disruption of cells. A severe conformational alteration or a change in the identity of the donating heme is discussed. Redox titrations of the flash-induced absorption changes performed on whole cells in the presence of mediators yielded the following redox midpoint potentials: P798, Em = +240 mV; heme c553, Em = +190, +170, and +90 mV for the heme components oxidized after the first, second, and third flash, respectively. The results demonstrate that the pool of c553 hemes donating electrons to the RC is heterogeneous and that it consists of either several distinguishable cytochromes or multiheme cytochromes or both. The number of hemes reduced and the kinetics of heme rereduction after flash-induced oxidation were found to depend strongly on the degree of anaerobicity in the interior compartment of the cell. A model rationalizing the obtained results in terms of a set of differing redox components is proposed.

Time-resolved absorption and magnetic circular dichroism spectroscopy of cytochrome c3 from Desulfovibrio.

The UV-visible absorption and magnetic circular dichroism (MCD) spectra of the ferric, ferrous, CO-ligated forms and kinetic photolysis intermediates of the tetraheme electron-transfer protein cytochrome c3 (Cc3) are reported. Consistent with bis-histidinyl axial coordination of the hemes in this Class III c-type cytochrome, the Soret and visible region MCD spectra of ferric and ferrous Cc3 are very similar to those of other bis-histidine axially coordinated hemeproteins such as cytochrome b5. The MCD spectra indicate low spin state for both the ferric (S = 1/2) and ferrous (S = 0) oxidation states. CO replaces histidine as the axial sixth ligand at each heme site, forming a low-spin complex with an MCD spectrum similar to that of myoglobin-CO. Photodissociation of Cc3-CO (observed photolysis yield = 30%) produces a transient five-coordinate, high-spin (S = 2) species with an MCD spectrum similar to deoxymyoglobin. The recombination kinetics of CO with heme Fe are complex and appear to involve at least five first-order or pseudo first-order rate processes, corresponding to time constants of 5.7 microseconds, 62 microseconds, 425 microseconds, 2.9 ms, and a time constant greater than 1 s. The observed rate constants were insensitive to variation of the actinic photon flux, suggesting noncooperative heme-CO rebinding. The growing in of an MCD signal characteristic of bis-histidine axial ligation within tens of microseconds after photodissociation shows that, although heme-CO binding is thermodynamically favored at 1 atm CO, binding of histidine to the sixth axial site competes kinetically with CO rebinding.